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WO2025076367A1 - Récepteurs d'interféron-gamma (ifn-g) modifiés (ifngr) pour améliorer l'immunothérapie cellulaire - Google Patents

Récepteurs d'interféron-gamma (ifn-g) modifiés (ifngr) pour améliorer l'immunothérapie cellulaire Download PDF

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WO2025076367A1
WO2025076367A1 PCT/US2024/049981 US2024049981W WO2025076367A1 WO 2025076367 A1 WO2025076367 A1 WO 2025076367A1 US 2024049981 W US2024049981 W US 2024049981W WO 2025076367 A1 WO2025076367 A1 WO 2025076367A1
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seq
residues
receptor
signaling domain
intracellular signaling
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Eric TRAN
Myungkyu JANG
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Providence Health and Services Oregon
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4244—Enzymes
    • A61K40/4253—GTPases, e.g. Ras or Rho
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7156—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • FIELD This disclosure concerns chimeric polypeptides that include an interferon-gamma receptor (IFNGR) extracellular domain, a transmembrane domain and a heterologous intracellular signaling domain, immune cells expressing such chimeric polypeptides, and their use for cancer immunotherapy.
  • IFNGR interferon-gamma receptor
  • IFN- ⁇ Interferon-gamma
  • T cells which produce and secrete IFN- ⁇ upon recognition of antigen.
  • NK Natural killer
  • NKT cells can also produce IFN- ⁇ . This cytokine can directly act on tumor cells to inhibit cell proliferation and induce cell death upon binding its cognate receptor which is comprised of IFN- ⁇ receptor 1 (IFNGR1) and IFNGR2.
  • IFN- ⁇ can also upregulate major histocompatibility complex (MHC) molecules on tumor cells and antigen presenting cells (APCs) to enhance antitumor T-cell responses.
  • MHC major histocompatibility complex
  • APCs antigen presenting cells
  • IFNGR signaling in T cells can also inhibit their proliferation and promote cell death.
  • modified IFNGRs e.g., IFNG chimeric receptors
  • the IFNG chimeric receptors include the extracellular domain of IFNGR1 and/or IFNGR2 fused to the intracellular domains of other cytokine receptors, such as the interleukin-2 receptor, which promotes T cell activity.
  • IFNG chimeric receptors Upon activation, immune cells expressing the IFNG chimeric receptors secrete IFN- ⁇ , which binds to the IFNG chimeric receptors and transmits signals that enhance the proliferation and/or function of the engineered immune cells.
  • engineered immune cells can be used, for example, to enhance cancer immunotherapy.
  • nucleic acid molecules that encode a first chimeric polypeptide that includes an interferon gamma receptor 1 (IFNGR1) extracellular domain, a first transmembrane domain, and a first intracellular signaling domain.
  • the first intracellular signaling domain is a heterologous 6727-109858-02 signaling domain of a cytokine receptor.
  • the first intracellular signaling domain is a modified IFNGR1 intracellular signaling domain.
  • nucleic acid molecules that encode a first chimeric polypeptide as described above and further encode a second chimeric polypeptide that includes an interferon gamma receptor 2 (IFNGR2) extracellular domain, a second transmembrane domain, and a second intracellular signaling domain.
  • IFNGR2 interferon gamma receptor 2
  • the second intracellular signaling domain is a heterologous signaling domain of a cytokine receptor.
  • the second intracellular signaling domain is an IFNGR2 intracellular signaling domain, such as a wild-type IFNGR2 intracellular signaling domain.
  • first chimeric polypeptide and the second chimeric polypeptide are encoded by a single nucleic acid molecule and the coding sequence for the first chimeric polypeptide and the coding sequence for the second chimeric polypeptide are separated by a nucleic acid sequence encoding a self-cleaving peptide.
  • first chimeric polypeptide is encoded by a first nucleic acid molecule and the second chimeric polypeptide is encoded by a second nucleic acid molecule.
  • the first and/or second intracellular signaling domains are heterologous intracellular signaling domains from a cytokine receptor.
  • cytokine receptors include, but are not limited to, erythropoietin receptor (EPOR), growth hormone receptor (GHR), thrombopoietin receptor (TPOR), prolactin receptor (PRLR), leptin receptor (LEPR), thymic stromal lymphopoietin receptor (TSLPR), colony stimulating factor 2 receptor subunit beta (CSF2RB), interleukin 3 receptor subunit alpha (IL3RA), interleukin 2 receptor subunit beta (IL2RB), interleukin 7 receptor (IL7R), interleukin 9 receptor (IL9R), insulin receptor (INSR), membrane glycoprotein 130 (GP130), CD28, 4-1BB, inducible T cell costimulator (ICOS), CD2, toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), toll-like receptor 6 (TLR6), epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (I
  • the first intracellular signaling domain is a modified IFNGR1 signaling domain and/or the second intracellular signaling domain is a wild-type IFNGR2 signaling domain.
  • the transmembrane domain of the first chimeric polypeptide is a heterologous transmembrane domain (a transmembrane domain that is not from IFNGR1), such as a transmembrane domain from the same cytokine receptor as the first intracellular signaling domain and/or the transmembrane domain of the second chimeric polypeptide is a heterologous transmembrane domain (a transmembrane domain that is not from IFNGR2), such as a transmembrane domain from the same cytokine receptor as the second intracellular signaling domain.
  • the transmembrane domain of the first chimeric 6727-109858-02 polypeptide is an IFNGR1 transmembrane domain and/or the transmembrane domain of the second chimeric polypeptide is an IFNGR2 transmembrane domain.
  • the nucleic acid molecules are codon-optimized for expression in mammalian cells, such as human cells. Also provided herein are vectors that include a disclosed nucleic acid molecule or molecules and modified immune cells that include a nucleic acid molecule(s) or vector disclosed herein.
  • modified immune cells that express a first chimeric polypeptide that includes an IFNGR1 extracellular domain, a first transmembrane domain, and a first intracellular signaling domain.
  • the first intracellular signaling domain is a heterologous signaling domain of a cytokine receptor, such as signaling domain of any of the cytokine receptors listed above.
  • the first intracellular signaling domain is a modified IFNGR1 intracellular signaling domain.
  • modified immune cells that express a first chimeric polypeptide as described above and further express a second chimeric polypeptide that includes an IFNGR2 extracellular domain, a second transmembrane domain, and a second intracellular signaling domain.
  • the second intracellular signaling domain is a heterologous signaling domain of a cytokine receptor, such as signaling domain of any of the cytokine receptors listed above.
  • the second intracellular signaling domain is an IFNGR2 intracellular signaling domain, for example a wild-type IFNGR2 intracellular signaling domain.
  • the transmembrane domain of the first chimeric polypeptide is a heterologous transmembrane domain (a transmembrane domain that is not from IFNGR1), such as a transmembrane domain from the same cytokine receptor as the first intracellular signaling domain and/or the transmembrane domain of the second chimeric polypeptide is a heterologous transmembrane domain (a transmembrane domain that is not from IFNGR2), such as a transmembrane domain from the same cytokine receptor as the second intracellular signaling domain.
  • the transmembrane domain of the first chimeric polypeptide is an IFNGR1 transmembrane domain and/or the transmembrane domain of the second chimeric polypeptide is an IFNGR2 transmembrane domain.
  • pharmaceutical compositions that include a modified immune cell disclosed herein and a pharmaceutically acceptable carrier.
  • methods of treating cancer in a subject includes administering to the subject a therapeutically effective amount of a modified immune cell or pharmaceutical composition disclosed herein.
  • the method further includes administering one or more additional anti-cancer therapies to the subject.
  • FIG.1 General schematic of wild-type (i) and exemplary chimeric IFNGRs (ii-v).
  • the IFNGR is comprised of two homodimers of IFNGR1 (R1) and IFNGR2 (R2) that are activated upon binding to IFN- ⁇ .
  • Chimeric IFNGRs include extracellular domains from the IFNGR fused to cytoplasmic signaling domains from other receptors that can activate pathways that enhance T-cell activity. Entire cytoplasmic domains (e.g., ii, iii, iv) or parts of cytoplasmic domains (e.g., v) from other receptors can be used.
  • the transmembrane domain can be derived from any receptor (e.g., from the IFNGR or the signaling receptor).
  • FIGS.2A-2C Biochemical signaling of IFNG chimeric receptors. T cells were transduced with a retroviral vector encoding the KRAS G12D-reactive TCR alone (FIG.2A), and co-transduced with either a retroviral vector encoding an IFNG chimeric receptor containing the IFNGR1 extracellular domain fused to the IL-2 receptor beta chain transmembrane and cytoplasmic domains and the IFNGR2 extracellular domain fused to the common gamma chain transmembrane and cytoplasmic domains (IFNGR/IL2R) (FIG.2B), or a retroviral vector encoding an IFNG receptor containing the IFNGR1 extracellular domain fused to the common beta chain cytoplasmic and transmembrane domains and the IFNGR2 extracellular domain fused to the G
  • STAT5 is a key transcription factor involved in IL2R and GMCSFR signaling.
  • cells were washed, and rested overnight in media without exogenous cytokines. Cells were then stimulated with nothing (no stim), 300 IU/ml IL-2 (positive control), or titrating doses of IFNG. Thirty minutes after stimulation, cells were fixed and permeabilized with cold methanol and stained with antibodies specific for phosphorylated STAT5 (P-STAT5). Cells were washed and analyzed by flow cytometry. Data were gated on CD8+mTCRb+ transduced T cells.
  • FIGS.3A-3C In vitro cytotoxicity of chimeric IFNG receptors against a pancreatic cancer cell line. T cells were transduced to express the KRAS G12D-reactive TCR alone (FIG.3A), and co-transduced to express either an IFNG chimeric receptor containing the IFNGR1 extracellular domain fused to the IL-2 receptor beta chain transmembrane and cytoplasmic domains and the IFNGR2 extracellular domain fused to the common gamma chain transmembrane and cytoplasmic domains (IFNGR/IL2R) (FIG.3B), or an IFNG receptor containing the IFNGR1 extracellular domain fused to the common beta chain cytoplasmic and transmembrane domains and the IFNGR2 extracellular domain fused to the GMCSF receptor alpha transmembrane and cytoplasmic domains (IFNGR/GMCSFR) (FIG.3C).
  • IFNGR/GMCSFR GMCSF receptor alpha trans
  • T cells then underwent a “serial kill” assay in which the T cells were initially cocultured at 5:1 E:T (50,000 T cells, 10,000 tumor cells) with the HPAC pancreatic cancer cell line that was genetically engineered to express GFP. An additional 10,000 HPAC-GFP cells were added about every 2 days, 5 times total, until the end of the assay.
  • FIG.3A GFP fluorescence (as a measure of tumor cell growth) of the KRAS-G12D-positive and HLA- C*08:02-positive HPAC-GFP pancreatic cancer cell line in the presence of T cells transduced to express the HLA-C*08:02-restricted KRAS-G12D reactive TCR without (circles) or with 300 IU/ml IL-2 (squares).
  • FIGS.3B-3C GFP fluorescence of the KRAS-G12D-positive and HLA-C*08:02-positive HPAC-GFP 6727-109858-02 pancreatic cancer cell line in the presence of T cells co-transduced to express the HLA-C*08:02-restricted KRAS-G12D reactive TCR and IFNGR/IL2R chimeric receptors (FIG.3B, triangles) or IFNGR/GMCSFR chimeric receptors (FIG.3C, triangles). No exogenous cytokines were added to the coculture.
  • SEQ ID NO: 147 is an exemplary amino acid sequence of wild-type human IFNGR1 (GENBANK Accession No. NP_000407.1).
  • a polypeptide includes singular or plural polypeptides and can be considered equivalent to the phrase “at least one polypeptide.”
  • the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
  • 2A peptide A type of self-cleaving peptide encoded by some RNA viruses, such as picornaviruses.
  • 2A peptides function by making the ribosome skip the synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the downstream peptide (Kim et al., PLoS One 6(4):e18556, 2011).
  • the "cleavage” occurs between the glycine and proline residues found on the C-terminus of the 2A peptide.
  • Exemplary 2A peptides include, but are not limited to, the 2A peptides encoded by Thosea asigna virus (TaV), equine rhinitis A virus (ERAV), porcine teschovirus-1 (PTV1) and foot and mouth disease virus (FMDV), which are set forth herein as SEQ ID NOs: 149-152 and listed below: P2A: ATNFSLLKQAGDVEENPGP (SEQ ID NO: 149) F2A: VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 150) E2A: QCTNYALLKLAGDVESNPGP (SEQ ID NO: 151) T2A: EGRGSLLTCGDVEENPGP (SEQ ID NO: 152) 4-1BB: A member of the TNF-receptor family.4-1BB is involved in T cell clonal expansion, survival, and development.
  • TaV asigna virus
  • EAV equine rhinitis A virus
  • PTV1 porcine
  • This protein can also induce proliferation in peripheral monocytes, enhance T cell apoptosis, and regulate Th1 cell responses.
  • 4-1BB is also known as TNFRSF9, ILA, and CD137.
  • Nucleic acid and amino acid sequences for human 4-1BB (and homologs thereof) are publicly available, such as under NCBI Gene ID 3604.
  • Autologous Refers to tissues, cells or nucleic acids taken from an individual’s own tissues. For example, in an autologous transfer or transplantation of modified T cells described herein, the donor and recipient are the same person. Therapies utilizing engineered autologous T cells are patient-specific, as the therapeutic T cells are created from a patient’s own cells.
  • PBMCs peripheral blood mononuclear cells
  • CD8+ T cells are isolated from the collected PBMCs, activated, and transduced with a nucleic acid encoding a chimeric IFNGR polypeptide(s) to produce the autologous T cells, which may then be culture-expanded, formulated, and optionally cryopreserved.
  • Cancer A malignant tumor characterized by abnormal or uncontrolled cell growth.
  • Methodastatic disease refers to cancer cells that have left the original tumor site and migrated to other parts of the body, for example via the bloodstream or lymph system.
  • CD2 A surface antigen found on peripheral blood T-cells. This protein optimizes immune recognition. CD2 is also known as LFA-2. Nucleic acid and amino acid sequences for human CD2 (and homologs thereof) are publicly available, such as under NCBI Gene ID 914.
  • CD28 A protein important for T-cell proliferation and survival, cytokine production, and T-helper type-2 development. Nucleic acid and amino acid sequences for human CD28 (and homologs thereof) are publicly available, such as under NCBI Gene ID 940. As used herein, “CD28_YMFM” refers to a modified CD28 reported to be more active than WT CD28 (the sequence of the CD28_YMFM intracellular signaling domain is set forth herein as residues 273-313 of SEQ ID NO: 16).
  • CSF2RB is also known as CD131, IL3RB, and IL5RB. Nucleic acid and amino acid sequences for human CSF2RB (and homologs thereof) are publicly available, such as under NCBI Gene ID 1439.
  • Ectopic The term “ectopic,” as applied to molecules herein (for example, IFNGR proteins, TCRs, and polynucleotides encoding the same), refers to a polypeptide/protein or polynucleotide in a specific environment or context in which it is not normally present. For example, if a host cell is transformed with a polynucleotide that does not occur in the untransformed host cell in nature, then that polynucleotide is ectopic to the host cell.
  • ectopic proteins herein specifically include proteins that are identical in amino acid sequence to a protein already present in a host cell, but that are expressed in a different cellular context than the protein with the same sequence already present in the host cell; for example, wherein the ectopic protein is constitutively expressed or expressed under different conditions.
  • ectopic polynucleotides encoding the proteins herein specifically include polynucleotides that are identical in sequence to those already present in a host cell, but that are located in a different cellular or genomic context than the polynucleotide with the same sequence already present in the host cell.
  • a polynucleotide that is located in a different location in the host cell than a polynucleotide with the same sequence is normally integrated in the of the host cell (for example, as a component of an expression construct integrated at a different genomic locus than the polynucleotide of the same sequence is normally found) is ectopic to the host cell.
  • a polynucleotide that is present in a plasmid or vector in the host cell is ectopic to the host cell when a polynucleotide with the same sequence is only normally present in the genome of the host cell.
  • Epidermal growth factor receptor A member of the protein kinase superfamily. EGFR is a transmembrane glycoprotein that binds to epidermal growth factor (EGF), causing a tyrosine 6727-109858-02 autophosphorylation cascade and cell proliferation. EGFR is also known as ERBB, ERBB1 and HER1.
  • EPOR Erythropoietin receptor
  • Jak2 tyrosine kinase which activates different intracellular pathways including Ras/MAP kinase, phosphatidylinositol 3-kinase and STAT transcription factors.
  • Nucleic acid and amino acid sequences for human EPOR are publicly available, such as under NCBI Gene ID 2057.
  • EPORtr refers to a truncated EPOR lacking the C-terminal 70 amino acids. EPORtr has been reported to be more active than WT EPOR.
  • the amino acid sequence of EPORtr intracellular domain is set forth herein as residues 269-433 of SEQ ID NO: 2.
  • expression As used herein, “expression” of a polynucleotide refers to the process by which the coded information of a transcriptional unit is converted into a polypeptide. The expression of a coding sequence can be influenced by external signals; for example, exposure of a cell, tissue, or organism to a signal (for example, an agonist or antigen that binds a cellular receptor) that increases or decreases gene expression.
  • Glycoprotein 130 A signal transducer shared by many cytokines, including interleukin 6 (IL6), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and oncostatin M (OSM). This protein functions as a part of the cytokine receptor complex. The activation of GP130 is dependent upon the binding of cytokines to their receptors.
  • GP130 is also known as interleukin 6 cytokine family signal transducer (IL6ST) and CD130. Nucleic acid and amino acid sequences for human GP130 (and homologs thereof) are publicly available, such as under NCBI Gene ID 3572.
  • Granulocyte-macrophage colony stimulating factor receptor alpha (GMCSFRA): The alpha subunit of the heterodimeric receptor for colony stimulating factor 2, a cytokine which controls the production, differentiation, and function of granulocytes and macrophages. This protein is a member of the cytokine family of receptors.
  • GMCSFRA is also known as colony stimulating factor 2 receptor subunit alpha (CSF2RA) and CD116.
  • GHR Growth hormone receptor
  • GHR A member of the type I cytokine receptor family, which is a transmembrane receptor for growth hormone. Binding of growth hormone to the receptor leads to activation of an intra- and intercellular signal transduction pathway leading to growth. GHR is also known as GHBP and GHIP. Nucleic acid and amino acid sequences for human GHR (and homologs thereof) are publicly available, such as under NCBI Gene ID 2690.
  • Heterologous The term “heterologous,” as applied to polypeptides and/or polynucleotides herein, means of different origin.
  • a host cell is transformed with a polynucleotide that does not occur in the untransformed host cell in nature, then that polynucleotide is heterologous (and ectopic) to the 6727-109858-02 host cell.
  • a host cell expresses a polypeptide (e.g., IFNGR chimeric polypeptides) that does not occur in the host cell in nature, then that polypeptide is heterologous (and ectopic) to the host cell.
  • chimeric polypeptides comprised of two or more components can include heterologous sequences, for example a transmembrane domain or intracellular signaling domain are heterologous to an IFNGR1 extracellular domain if they are from proteins other than IFNGR1.
  • different elements e.g., promoters, enhancers, coding sequences, and terminators
  • heterologous polynucleotides herein also specifically include a polynucleotide that is identical in sequence to a polynucleotide already present in a host cell, but that is linked to a different regulatory sequence and/or is present at a different copy number in the host cell.
  • Inducible T cell costimulator A member of the CD28 and CTLA-4 cell surface receptor family.
  • the ICOS protein forms homodimers which are involved in cell-cell signaling, immune response, and regulation of cell proliferation.
  • ICOS is also known as CD278.
  • Nucleic acid and amino acid sequences for human ICOS (and homologs thereof) are publicly available, such as under NCBI Gene ID 29851.
  • Inhibiting, ameliorating, or treating a disease “Inhibiting” a condition refers to inhibiting the full development of a condition or disease, for example, cancer or a tumor. Inhibition of a condition occurs within the spectrum from partial inhibition to substantially complete inhibition of the disease.
  • the term “inhibiting” refers to reducing or delaying the onset or progression of a condition. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer. “Treating” refers to a therapeutic intervention that decreases or inhibits a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor size or tumor burden.
  • a subject to be administered an effective amount of the disclosed modified immune cells can be identified by standard diagnosing techniques for such a disorder, for example, presence of the disease or disorder or risk factors to develop the disease or disorder.
  • Insulin-like growth factor 1 receptor This receptor binds insulin-like growth factor and has tyrosine kinase activity. IGF1R is also known as IGFR, CD221 and JTK13. Nucleic acid and amino acid sequences for human IGF1R (and homologs thereof) are publicly available, such as under NCBI Gene ID 3480.
  • Insulin receptor (INSR) A member of the receptor tyrosine kinase family of proteins. Binding of insulin or other ligands to this receptor activates the insulin signaling pathway, which regulates glucose uptake and release, as well as the synthesis and storage of carbohydrates, lipids and protein. INSR is also known as CD220.
  • Nucleic acid and amino acid sequences for human INSR are publicly available, such as under NCBI Gene ID 3643.
  • Interferon gamma receptor 1 IFNGR1
  • the human interferon gamma receptor is a heterodimer of IFNGR1 and IFNGR2.
  • IFNGR1 is also known as CD119.
  • Nucleic acid and amino acid sequences for human IFNGR1 are publicly available, such as under NCBI Gene ID 3459.
  • An exemplary wild-type 6727-109858-02 human IFNGR1 amino acid sequence is set forth herein as SEQ ID NO: 147.
  • IFNGR1 with a modified intracellular domain are disclosed herein as follows: Modified IFNGR1 Descriptor IFNGR1-2RB-S5 The IFNGR1 STAT1 binding site (FGYDKPH; residues 455-461 of m s h of 34 Interferon gamma receptor 2 (IFNGR2): The non-ligand binding chain (beta) of the gamma interferon receptor.
  • IFNGR2 Interferon gamma receptor 2
  • the human interferon gamma receptor is a heterodimer of IFNGR1 and IFNGR2. Nucleic acid and amino acid sequences for human IFNGR2 (and homologs thereof) are publicly available, such as under NCBI Gene ID 3460.
  • Interleukin 2 receptor subunit beta (IL2RB): The beta subunit of the interleukin 2 receptor.
  • IL2RB is a type I membrane protein.
  • the interleukin 2 receptor which is involved in T cell-mediated immune responses, is present in 3 forms with respect to ability to bind interleukin 2.
  • the low affinity form is a monomer of the alpha subunit, the intermediate affinity form consists of an alpha/beta subunit heterodimer, and the high affinity form consists of an alpha/beta/gamma subunit heterotrimer.
  • IL2RB is also known as CD122 and IL15RB. Nucleic acid and amino acid sequences for human IL2RB (and homologs thereof) are publicly available, such as under NCBI Gene ID 3560.
  • Interleukin 2 receptor subunit gamma IL2RG: A protein that is the signaling component of many interleukin receptors, including the receptors for IL-2, -4, -7 and -21, and is thus referred to as the common gamma chain. IL2RG is also known as CD132.
  • Interleukin 3 receptor subunit alpha (IL3RA): The ligand-binding subunit of the IL-3 receptor complex. IL3RA heterodimerizes with the common beta chain. Nucleic acid and amino acid sequences for human IL3RA (and homologs thereof) are publicly available, such as under NCBI Gene ID 3563.
  • Interleukin 6 receptor (IL6R or IL6RA): A subunit of the interleukin 6 (IL6) receptor complex. Interleukin 6 is a pleiotropic cytokine that regulates cell growth and differentiation and plays an important role in immune response.
  • Interleukin 7 receptor (IL7R or IL7RA): A receptor for interleukin 7 (IL7), which heterodimerizes with the common gamma chain to bind IL-7. IL7R is also known as CD127, IL7RA, and IL-7R-alpha. Nucleic acid and amino acid sequences for human IL7R (and homologs thereof) are publicly available, such as under NCBI Gene ID 3575.
  • Interleukin 9 receptor (IL9R): A cytokine receptor that mediates the effects of interleukin 9 (IL9).
  • the IL9 receptor complex requires this protein as well as the interleukin 2 receptor, gamma (IL2RG), a common gamma subunit shared by the receptors of many different cytokines. IL9 binding to the receptor leads to the activation of various JAK-STAT pathways. IL9R is also known as CD129. Nucleic acid and amino acid sequences for human IL9R (and homologs thereof) are publicly available, such as under NCBI Gene ID 3581.
  • IL10RA is also known as CD210, IL10R and IL-10R1. Nucleic acid and amino acid sequences for human IL10RA (and homologs thereof) are publicly available, such as under NCBI Gene ID 3587.
  • Interleukin 10 receptor subunit beta (IL10RB) A member of the cytokine receptor family. This subunit is an accessory chain essential for the activation of the interleukin 10 receptor complex. Nucleic acid and amino acid sequences for human IL10RB (and homologs thereof) are publicly available, such as under NCBI Gene ID 3588.
  • Interleukin 11 receptor subunit alpha (IL11RA) A receptor for IL11 and a member of the hematopoietic cytokine receptor family.
  • IL20RA Interleukin 20 receptor subunit alpha
  • IL20RA is a subunit for the interleukin 20 receptor.
  • IL20RA and IL20RB form a heterodimeric receptor for IL20.
  • Nucleic acid and amino acid sequences for human IL10RA (and homologs thereof) are publicly available, such as under NCBI Gene ID 53832.
  • Interleukin 20 receptor subunit beta A subunit of the IL20 receptor.
  • IL20RA and IL20RB form a heterodimeric receptor for IL20.
  • Nucleic acid and amino acid sequences for human IL20RB (and homologs thereof) are publicly available, such as under NCBI Gene ID 53833.
  • Interleukin 22 receptor IL22R or IL22RA1: A receptor for IL22 that belongs to the class II cytokine receptor family.
  • the IL22 receptor is a protein complex that consists of IL22RA1 and IL10 receptor beta, a subunit that is also shared by the receptor complex for IL10.
  • Isolated An “isolated” biological component (such as a polynucleotide, protein, or cell) has been substantially separated, produced apart from, or purified away from other biological components (e.g., other cells, chromosomal and extra-chromosomal DNA and RNA, and proteins). Cells, polynucleotides, and proteins that have been isolated specifically include cells, nucleic acid molecules, and proteins purified by standard purification methods.
  • MET proto-oncogene receptor tyrosine kinase A member of the receptor tyrosine kinase family of proteins. Binding of its ligand, hepatocyte growth factor, induces dimerization and activation of the receptor, which plays a role in cell survival, embryogenesis, and cellular migration and invasion. MET is also known as HGFR and c-Met. Nucleic acid and amino acid sequences for human MET (and homologs thereof) are publicly available, such as under NCBI Gene ID 4233.
  • OSMRB Oncostatin M receptor
  • OSMRB protein heterodimerizes with interleukin 6 signal transducer (IL6ST) and transduces oncostatin M signaling events.
  • OSMRB is also known as OSMR, IL-3RB and PLCA1.
  • Nucleic acid and amino acid sequences for human OSMRB (and homologs thereof) are publicly available, such as under NCBI Gene ID 9180.
  • Pharmaceutically acceptable carriers The pharmaceutically acceptable carriers of use are known to those of ordinary skill in the art. Remington: The Science and Practice of Pharmacy, 22 nd ed., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents. In general, the nature of the carrier will depend on the particular mode of 6727-109858-02 administration being employed.
  • the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as in a selected quantity in a vial.
  • Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agent can selectively be dispensed).
  • Prolactin receptor (PRLR) A receptor for the anterior pituitary hormone, prolactin, and belongs to the type I cytokine receptor family. Nucleic acid and amino acid sequences for human PRLR (and homologs thereof) are publicly available, such as under NCBI Gene ID 5618. Purified: The term purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified nucleic acid, protein or cell preparation is one in which the nucleic acid, protein, or cell is more enriched than in its original environment.
  • a preparation is purified such that the nucleic acid, protein, or cells represent at least 50% of the total nucleic acid, protein, or cell content of the preparation.
  • Recombinant A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence (such as a chimeric nucleic acid). This artificial combination can be accomplished by chemical synthesis or by the manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.
  • a chimeric protein is encoded by a recombinant nucleic acid.
  • a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell.
  • the nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid, or the nucleic acid can be integrated into the host cell chromosome.
  • regulatory elements refer to nucleic acid elements that influence the timing and level/amount of transcription (or RNA processing or stability) of an operably linked polynucleotide. Particular regulatory elements may be located upstream and/or downstream of a polynucleotide operably linked thereto. Also, particular regulatory elements operably linked to a polynucleotide may be located on the associated complementary strand of a double-stranded nucleic acid molecule. Regulatory elements include, for example and without limitation, promoters, translation leaders, introns, enhancers, stem-loop structures, repressor binding sequences, termination sequences, and polyadenylation recognition sequences.
  • a polynucleotide is operably linked to a promoter, such that the polynucleotide is expressed in a host cell.
  • Specific promoters herein utilized include 6727-109858-02 “constitutive” promoters, which refers to a promoter that is active under most or all conditions in a host cell.
  • a polynucleotide is operably linked to an “inducible” promoter, which refers to a promoter wherein the rate of transcription increases in response to an inducing agent (for example, a signal molecule).
  • ROS1 proto-oncogene 1 receptor tyrosine kinase A protein belonging to the subfamily of tyrosine kinase insulin receptor genes.
  • ROS1 is a type I integral membrane protein that functions as a growth and differentiation factor receptor.
  • Nucleic acid and amino acid sequences for human ROS1 (and homologs thereof) are publicly available, such as under NCBI Gene ID 6098.
  • Subject Living multi-cellular vertebrate organisms, a category that includes human and non- human mammals, such as non-human primates, pigs, sheep, cows, dogs, cats, rodents, and the like. In some examples, the subject is a human.
  • T cell A white blood cell (lymphocyte) that is an important mediator of the immune response.
  • T cells include, but are not limited to, CD4 + T cells and CD8 + T cells.
  • a CD4 + T lymphocyte is an immune cell that carries a marker on its surface known as “cluster of differentiation 4” (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses.
  • CD8 + T cells carry the “cluster of differentiation 8” (CD8) marker.
  • Activated T cells can be detected by an increase in cell proliferation and/or expression of or secretion of one or more cytokines (such as IL-2, IL-4, IL-6, IFN ⁇ , or TNF ⁇ ).
  • cytokines such as IL-2, IL-4, IL-6, IFN ⁇ , or TNF ⁇ .
  • Activation of CD8+ T cells can also be detected by an increase in cytolytic activity in response to an antigen.
  • a “modified T cell” is a T cell transduced or transfected with a heterologous nucleic acid (such as one or more of the nucleic acids or vectors disclosed herein) or expressing one or more heterologous proteins (such as a chimeric IFNGR polypeptide).
  • a heterologous nucleic acid such as one or more of the nucleic acids or vectors disclosed herein
  • heterologous proteins such as a chimeric IFNGR polypeptide.
  • TPOR Thrombopoietin receptor
  • MPL thrombopoietin receptor
  • the TPOR protein is 635 amino acids in length and includes a transmembrane domain, two extracellular cytokine receptor domains and two intracellular cytokine receptor box motifs. Upon binding of thrombopoietin, TPOR dimerizes and leads to tyrosine phosphorylation of multiple proteins, including those in the JAK, STAT and MAPK families. TPOR is also known as CD110, myeloproliferative leukemia protein, MPLV, C-MPL, THPOR and THCYT2. Nucleic acid and amino acid sequences for human TPOR (and homologs thereof) are publicly available, such as under NCBI Gene ID 4352.
  • Thymic stromal lymphopoietin receptor A member of the type I cytokine receptor family that functions as a receptor for thymic stromal lymphopoietin (TSLP). TSLP binding activates STAT3, STAT5, and JAK2 pathways, which control processes such as cell proliferation and development of the hematopoietic system. TSLPR is also known as cytokine receptor like factor 2 (CRLF2). Nucleic acid and amino acid sequences for human TSLPR (and homologs thereof) are publicly available, such as under NCBI Gene ID 64109.
  • TLR1 A member of the toll-like receptor family, which plays a role in pathogen recognition and innate immunity. Infectious agents express pathogen-associated molecular patterns (PAMPs) that TLRs recognize and respond to by producing cytokines. TLR1 is also known as CD281. Nucleic acid and amino acid sequences for human TLR1 (and homologs thereof) are publicly available, such as under NCBI Gene ID 7096. Toll-like receptor 2 (TLR2): A member of the toll-like receptor family which is involved in pathogen recognition and activation of innate immunity. TLR2 is also known as CD282.
  • TLR4 A member of the toll-like receptor family which is involved in pathogen recognition and activation of innate immunity. This receptor has been implicated in signal transduction events induced by lipopolysaccharide. TLR4 is also known as CD284. Nucleic acid and amino acid sequences for human TLR4 (and homologs thereof) are publicly available, such as under NCBI Gene ID 7099.
  • TLR6 A member of the toll-like receptor family which is involved in pathogen recognition and activation of innate immunity.
  • TLR6 is also known as CD286. Nucleic acid and amino acid sequences for human TLR6 (and homologs thereof) are publicly available, such as under NCBI Gene ID 10333. Transformation/Transduction: As used herein, the terms “transformation” and “transduction” are used interchangeably to refer to the transfer of one or more polynucleotide(s) into a cell.
  • a cell is “transformed” or “transduced” by a polynucleotide when a nucleic acid molecule comprising the polynucleotide is introduced into the cell, and the polynucleotide becomes stably replicated by the cell, either by incorporation of the polynucleotide into the cellular genome, or by episomal replication.
  • the transformation and transduction encompass all techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to, transduction with viral vectors, electroporation, microinjection, direct DNA uptake, and microprojectile bombardment.
  • Vector Nucleic acid molecules as introduced into a cell, for example, to produce a transformed cell.
  • a vector may include genetic elements that permit it to replicate in the host cell.
  • vectors include but are not limited to plasmids, viruses, and linear DNA molecules that carry ectopic nucleic acids into a cell under suitable conditions.
  • the vector is a virus vector, such as a lentivirus vector.
  • the present disclosure describes chimeric interferon gamma receptors (IFNGRs) that include an extracellular domain from IFNGR1, a transmembrane domain (from IFNGR1 or a heterologous protein), and an intracellular signaling domain.
  • IFNGRs chimeric interferon gamma receptors
  • the intracellular signaling domain can be from a heterologous cytokine 6727-109858-02 receptor or can be a modified IFNGR1 intracellular signaling domain.
  • chimeric IFNGRs that include an extracellular domain from IFNGR2, a transmembrane domain (from IFNGR2 or a heterologous protein), and an intracellular signaling domain from a heterologous cytokine receptor (or in some instances, from wild-type IFNGR2).
  • the present disclosure also describes nucleic acid molecules that encode a chimeric IFNGR1 polypeptide. In some aspects, the nucleic acid molecule encodes both a chimeric IFNGR1 polypeptide and a chimeric IFNGR2 polypeptide.
  • a first nucleic acid molecule encodes a chimeric IFNGR1 polypeptide and a second nucleic acid molecule encodes a chimeric IFNGR2 polypeptide.
  • modified immune cells such as T cells
  • modified immune cells that include a nucleic acid molecule or molecules disclosed herein, as well as modified immune cells that express a chimeric IFNGR1 polypeptide and/or a chimeric IFNGR2 polypeptide.
  • Use of the modified immune cells such as for the treatment of cancer, is also described.
  • the modified immune cells can also be used to enhance the effectiveness of other immunotherapies or anti-cancer treatments in a subject in need thereof.
  • nucleic acid molecules that encode a first chimeric polypeptide that includes an interferon gamma receptor 1 (IFNGR1) extracellular domain, a first transmembrane domain, and a first intracellular signaling domain.
  • the first intracellular signaling domain is a heterologous signaling domain of a cytokine receptor.
  • the first intracellular signaling domain is a modified IFNGR1 intracellular signaling domain.
  • IFNGR2 interferon gamma receptor 2
  • the second intracellular signaling domain is a heterologous signaling domain of a cytokine receptor. In other aspects, the second intracellular signaling domain is an IFNGR2 intracellular signaling domain.
  • the first chimeric polypeptide and the second chimeric polypeptide are encoded by a single nucleic acid molecule and the coding sequence for the first chimeric polypeptide and the coding sequence for the second chimeric polypeptide are separated by a nucleic acid sequence encoding a self-cleaving peptide.
  • the first chimeric polypeptide is encoded by a first nucleic acid molecule and the second chimeric polypeptide is encoded by a second nucleic acid molecule.
  • the first intracellular signaling domain is a heterologous intracellular signaling domain of a first cytokine receptor and/or the second intracellular signaling domain is a heterologous intracellular signaling domain of a second cytokine receptor.
  • the first cytokine receptor and the second cytokine receptor are individually selected from the group consisting of: erythropoietin receptor (EPOR), growth hormone receptor (GHR), thrombopoietin receptor (TPOR), prolactin receptor (PRLR), leptin receptor (LEPR), thymic stromal lymphopoietin receptor (TSLPR), colony stimulating factor 2 receptor subunit beta (CSF2RB), interleukin 3 receptor subunit alpha (IL3RA), interleukin 2 receptor subunit beta (IL2RB), interleukin 7 receptor (IL7R), interleukin 9 receptor (IL9R), insulin receptor (INSR), membrane glycoprotein 130 (GP130), CD28, 4-1BB, inducible
  • the amino acid sequence of the first intracellular signaling domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 269-503 of SEQ ID NO: 1; residues 269-433 of SEQ ID NO: 2; residues 270-619 of SEQ ID NO: 3; residues 268-389 of SEQ ID NO: 4; residues 270-633 of SEQ ID NO: 5; residues 269-571 of SEQ ID NO: 6; residues 267-385 of SEQ ID NO: 7; residues 263-699 of SEQ ID NO: 8; residues 266-318 of SEQ ID NO: 9; residues 271-555 of SEQ ID NO: 10; residues 271- 465 of SEQ ID NO: 11; residues 267-496 of SEQ ID NO: 12; residues 269-671 of SEQ ID NO: 13; residues 268-544 of SEQ
  • the amino acid sequence of the first intracellular signaling domain comprises or consists of residues 269-503 of SEQ ID NO: 1; residues 269-433 of SEQ ID NO: 2; residues 270-619 of SEQ ID NO: 3; residues 268-389 of SEQ ID NO: 4; residues 270-633 of SEQ ID NO: 5; residues 269-571 of SEQ ID NO: 6; residues 267-385 of SEQ ID NO: 7; residues 263-699 of SEQ ID 6727-109858-02 NO: 8; residues 266-318 of SEQ ID NO: 9; residues 271-555 of SEQ ID NO: 10; residues 271-465 of SEQ ID NO: 11; residues 267-496 of SEQ ID NO: 12; residues 269-671 of SEQ ID NO: 13; residues 268-544 of SEQ ID NO: 14; residues 281-313 of SEQ ID NO: 15; residues 273-313 of SEQ ID NO: 16; residues 2
  • the amino acid sequence of the second intracellular signaling domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 801-1035 of SEQ ID NO: 28; residues 731-895 of SEQ ID NO: 29; residues 918-1267 of SEQ ID NO: 30; residues 686-807 of SEQ ID NO: 31; residues 932- 1295 of SEQ ID NO: 32; residues 869-1171 of SEQ ID NO: 33; residues 610-1046 of SEQ ID NO: 34; residues 685-879 of SEQ ID NO: 35; residues 969-1371 of SEQ ID NO: 36; residues 1000-1053 of SEQ ID NO: 37; residues 851-936 of SEQ ID NO: 38; residues 761-846 of SEQ ID NO: 39; residues 792-877 of SEQ ID NO: 40; residues 841-1117 of SEQ ID NO: 28; residues 731-8
  • the amino acid sequence of the second intracellular signaling domain comprises or consists of residues 801-1035 of SEQ ID NO: 28; residues 731-895 of SEQ ID NO: 29; residues 918-1267 of SEQ ID NO: 30; residues 686-807 of SEQ ID NO: 31; residues 932-1295 of SEQ ID NO: 32; residues 869-1171 of SEQ ID NO: 33; residues 610-1046 of SEQ ID NO: 34; residues 685-879 of SEQ ID NO: 35; residues 969-1371 of SEQ ID NO: 36; residues 1000-1053 of SEQ ID NO: 37; residues 851-936 of SEQ ID NO: 38; residues 761-846 of SEQ ID NO: 39; residues 792-877 of SEQ ID NO: 40; residues 841-1117 of SEQ ID NO: 41; residues 615-655 of SEQ ID NO: 42; residues 615-655 of SEQ ID NO: 43; residues 616-657 of S
  • the first intracellular signaling domain is a modified IFNGR1 signaling domain; and/or the first intracellular signaling domain is a modified IFNGR1 signaling domain and the second intracellular signaling domain is a wild-type IFNGR2 signaling domain.
  • the modified IFNGR1 signaling domain includes substitution of one or more amino acids that make up the IFNGR1 STAT1 binding site.
  • IFNGR1-2RB-S5 modified IFNGR1 referred to herein as IFNGR1-2RB-S5 (residues 267-489 of SEQ ID NO: 68), IFNGR1-2RB-S5- SOCSYF (residues 267-489 of SEQ ID NO: 69), IFNGR1-2RB-Cterm20 (residues 267-472 of SEQ ID NO: 70), IFNGR1-GHR-S5x2 (residues 267-489 of SEQ ID NO: 71), IFNGR1-GHR-Cyt45 (residues 267-497 of SEQ ID NO: 72), or IFNGR1-EPOR-S5 (residues 267-489 of SEQ ID NO: 73).
  • the amino acid sequence of the modified IFNGR1 intracellular signaling domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 267-489 of SEQ ID NO: 68; residues 267-489 of SEQ ID NO: 69; residues 267-472 of SEQ ID NO: 70; residues 267-489 of SEQ ID NO: 71; residues 267-497 of SEQ ID NO: 72; or residues 267-489 of SEQ ID NO: 73.
  • the amino acid sequence of the modified IFNGR1 intracellular signaling domain comprises or consists of residues 267-489 of SEQ ID NO: 68; residues 267-489 of SEQ ID NO: 69; residues 267-472 of SEQ ID NO: 70; residues 267-489 of SEQ ID NO: 71; residues 267-497 of SEQ ID NO: 72; or residues 267-489 of SEQ ID NO: 73.
  • the wild-type IFNGR2 intracellular signaling domain includes or consists of residues 288-356 of SEQ ID NO: 148.
  • the transmembrane domain of the first chimeric polypeptide is a heterologous transmembrane domain (a transmembrane domain that is not from IFNGR1), such as a transmembrane domain from the same cytokine receptor as the first intracellular signaling domain and/or the transmembrane domain of the second chimeric polypeptide is a heterologous transmembrane domain (a transmembrane domain that is not from IFNGR2), such as a transmembrane domain from the same cytokine receptor as the second intracellular signaling domain.
  • the transmembrane domain of the first chimeric polypeptide is an IFNGR1 transmembrane domain and/or the transmembrane domain of the second chimeric polypeptide is an IFNGR2 transmembrane domain.
  • the nucleic acid molecule encodes both the first and second chimeric polypeptides
  • the coding sequence for the first chimeric polypeptide and the coding sequence for the second chimeric polypeptide are separated by a nucleic acid sequence encoding a self-cleaving peptide.
  • the self-cleaving peptide is a 2A peptide.
  • the 2A peptide is a porcine teschovirus-12A peptide (P2A), a Thosea asigna virus 2A peptide (T2A), an equine rhinitis A virus 2A peptide (E2A), or a foot and mouth disease virus 2A peptide (F2A).
  • P2A porcine teschovirus-12A peptide
  • T2A Thosea asigna virus 2A peptide
  • E2A equine rhinitis A virus 2A peptide
  • F2A foot and mouth disease virus 2A peptide
  • the amino acid sequence of the P2A peptide includes SEQ ID NO: 149
  • the amino acid sequence of the F2A peptide includes SEQ ID NO: 150
  • the amino acid sequence of the E2A peptide includes SEQ ID NO: 151
  • the amino acid sequence of the T2A peptide includes SEQ ID NO: 152.
  • the amino acid sequence of the self-cleaving peptide includes residues 504-530 of SEQ ID NO: 28 (which includes a furin cleavage site and a spacer sequence) or residues 512-230 of SEQ ID NO: 28.
  • the nucleic acid molecules are codon-optimized for expression in mammalian cells, such as human cells.
  • the nucleic acid molecule encodes only a first chimeric polypeptide
  • the nucleic acid molecule includes the nucleotide sequence of any one of SEQ ID NOs: 74-100, or a degenerate variant thereof.
  • the nucleic acid molecule encodes both the first and second chimeric polypeptides
  • the nucleic acid molecule includes the nucleotide sequence of any one of SEQ ID NOs: 101-146, or a degenerate variant thereof.
  • the nucleic acid molecule is operably linked to a promoter.
  • vectors that include a nucleic acid molecule or molecules disclosed herein.
  • the vector is a viral vector, such as a gammaretroviral vector or a lentiviral vector.
  • modified immune cells that include a nucleic acid molecule or molecules or vector disclosed herein.
  • the immune cell is a T cell.
  • the T cell is a tumor-reactive T cell or a tumor-infiltrating lymphocyte (TIL).
  • TIL tumor-infiltrating lymphocyte
  • the immune cell is a B cell, NK cell, NKT cell, macrophage or dendritic cell.
  • the immune cell is a human immune cell.
  • modified immune cells that express a first chimeric polypeptide that includes an IFNGR1 extracellular domain, a first transmembrane domain, and a first intracellular signaling domain.
  • the first intracellular signaling domain is a heterologous signaling domain of a 6727-109858-02 cytokine receptor.
  • the first intracellular signaling domain is a modified IFNGR1 intracellular signaling domain.
  • modified immune cells that express a first chimeric polypeptide as described above and further express a second chimeric polypeptide that includes an IFNGR2 extracellular domain, a second transmembrane domain, and a second intracellular signaling domain.
  • the second intracellular signaling domain is a heterologous signaling domain of a cytokine receptor. In other aspects, the second intracellular signaling domain is an IFNGR2 intracellular signaling domain, for example a wild-type IFNGR2 intracellular signaling domain.
  • the first cytokine receptor and the second cytokine receptor are individually selected from the group consisting of EPOR, GHR, TPOR, PRLR, LEPR, TSLPR, CSF2RB, IL3RA, IL2RB, IL7R, IL9R, INSR, GP130, CD28, 4-1BB, ICOS, CD2, TLR1, TLR2, TLR4, TLR6, EGFR, IGF1R, MET, ROS1, GMCSFRA, IL2RG, IL6R, IL11RA, OSMRB, IL10RA, IL10RB, IL22R, IL20RA and IL20RB.
  • the amino acid sequence of the first intracellular signaling domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 269-503 of SEQ ID NO: 1; residues 269-433 of SEQ ID NO: 2; residues 270- 619 of SEQ ID NO: 3; residues 268-389 of SEQ ID NO: 4; residues 270-633 of SEQ ID NO: 5; residues 269-571 of SEQ ID NO: 6; residues 267-385 of SEQ ID NO: 7; residues 263-699 of SEQ ID NO: 8; residues 266-318 of SEQ ID NO: 9; residues 271-555 of SEQ ID NO: 10; residues 271-465 of SEQ ID NO: 11; residues 267-496 of SEQ ID NO: 12; residues 269-671 of SEQ ID NO: 13; residues 268-544 of SEQ ID NO: 14; residues 281-3
  • the amino acid sequence of the first intracellular signaling domain comprises or consists of residues 269-503 of SEQ ID NO: 1; residues 269-433 of SEQ ID NO: 2; residues 270-619 of SEQ ID NO: 3; residues 268-389 of SEQ ID NO: 4; residues 270- 633 of SEQ ID NO: 5; residues 269-571 of SEQ ID NO: 6; residues 267-385 of SEQ ID NO: 7; residues 263-699 of SEQ ID NO: 8; residues 266-318 of SEQ ID NO: 9; residues 271-555 of SEQ ID NO: 10; residues 271-465 of SEQ ID NO: 11; residues 267-496 of SEQ ID NO: 12; residues 269-671 of SEQ ID NO: 13; residues 268-544 of SEQ ID NO: 14; residues 281-313 of SEQ ID NO: 15; residues 273-313 of SEQ ID NO: 16;
  • the amino acid sequence of the second intracellular signaling domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 801-1035 of SEQ ID NO: 28; residues 731-895 of SEQ ID NO: 29; residues 918-1267 of SEQ ID NO: 30; residues 686-807 of SEQ ID NO: 31; residues 932-1295 of SEQ ID NO: 32; residues 869-1171 of SEQ ID NO: 33; residues 610-1046 of SEQ ID NO: 34; residues 685-879 of SEQ ID NO: 35; residues 969-1371 of SEQ ID NO: 36; residues 1000-1053 of SEQ ID NO: 37; residues 851-936 of SEQ ID NO: 38; residues 761-846 of SEQ ID NO: 39; residues 792-877 of SEQ ID NO: 40; residues 841-1117 of SEQ ID NO:
  • the amino acid sequence of the second intracellular signaling domain comprises or consists of residues 801- 1035 of SEQ ID NO: 28; residues 731-895 of SEQ ID NO: 29; residues 918-1267 of SEQ ID NO: 30; residues 686-807 of SEQ ID NO: 31; residues 932-1295 of SEQ ID NO: 32; residues 869-1171 of SEQ ID NO: 33; residues 610-1046 of SEQ ID NO: 34; residues 685-879 of SEQ ID NO: 35; residues 969-1371 of SEQ ID NO: 36; residues 1000-1053 of SEQ ID NO: 37; residues 851-936 of SEQ ID NO: 38; residues 761- 846 of SEQ ID NO: 39; residues 792-877 of SEQ ID NO: 40; residues 841-1117 of SEQ ID NO: 41; residues 615-655 of SEQ ID NO: 42; residues 615-655 of SEQ ID NO: 43; residues 616-657 of SEQ
  • the first intracellular signaling domain is a modified IFNGR1 signaling domain; and/or the first intracellular signaling domain is a modified IFNGR1 signaling domain and the second intracellular signaling domain is a wild-type IFNGR2 signaling domain.
  • the amino acid sequence of the modified IFNGR1 intracellular signaling domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 267-489 of SEQ ID NO: 68; residues 267-489 of SEQ ID NO: 69; residues 267-472 of SEQ ID NO: 70; residues 267-489 of SEQ ID NO: 71; residues 267-497 of SEQ ID NO: 72; or residues 267-489 of SEQ ID NO: 73.
  • the amino acid sequence of the modified IFNGR1 intracellular signaling domain comprises or consists of residues 267-489 of SEQ ID NO: 68; residues 267-489 of SEQ ID NO: 69; residues 267-472 of SEQ ID NO: 70; residues 267-489 of SEQ ID NO: 71; residues 267-497 of SEQ ID NO: 72; or residues 267-489 of SEQ ID NO: 73.
  • the wild-type IFNGR2 intracellular signaling domain includes residues 288-356 of SEQ ID NO: 148.
  • the transmembrane domain of the first chimeric polypeptide is a heterologous transmembrane domain (a transmembrane domain that is not from IFNGR1), such as a transmembrane domain from the same cytokine receptor as the first intracellular signaling domain
  • the transmembrane domain of the second chimeric polypeptide is a heterologous transmembrane 6727-109858-02 domain (a transmembrane domain that is not from IFNGR2), such as a transmembrane domain from the same cytokine receptor as the second intracellular signaling domain.
  • the transmembrane domain of the first chimeric polypeptide is an IFNGR1 transmembrane domain and/or the transmembrane domain of the second chimeric polypeptide is an IFNGR2 transmembrane domain.
  • the amino acid sequence of the first chimeric polypeptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO:
  • the amino acid sequence of the first chimeric polypeptide comprises or consists of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; residues 1-503 of SEQ ID NO: 28; residues 1-433 of SEQ ID NO: 29; residues 1-619 of SEQ ID NO:
  • the amino acid sequence of the second chimeric polypeptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 531-1035 of SEQ ID NO: 28; residues 461-895 of SEQ ID NO: 29; residues 645-1297 of SEQ ID NO: 30; residues 417-807 of SEQ ID NO: 31; residues 661-1295 of SEQ ID NO: 32; residues 599-1171 of SEQ ID NO: 33; residues 346-1046 of SEQ ID NO: 34; residues 413-879 of SEQ ID NO: 35; residues 699-1371 of SEQ ID NO: 36; residues 727-1053 of SEQ ID NO: 37; residues 535- 936 of SEQ ID NO: 38; residues 493-846 of SEQ ID NO: 39; residues 524-877 of SEQ ID NO: 40; residues 572-1117 of SEQ ID NO:
  • the amino acid sequence of the second chimeric polypeptide comprises or consists of residues 531-1035 of SEQ ID NO: 28; residues 461-895 of SEQ ID NO: 29; residues 645-1297 of SEQ ID NO: 30; residues 417-807 of SEQ ID NO: 31; residues 661-1295 of SEQ ID NO: 32; residues 599-1171 of SEQ ID NO: 33; residues 346-1046 of SEQ ID NO: 34; residues 413-879 of SEQ ID NO: 35; residues 699-1371 of SEQ ID NO: 36; residues 727-1053 of SEQ ID NO: 37; residues 535-936 of SEQ ID NO: 38; residues 493-846 of SEQ ID NO: 39; residues 524-877 of SEQ ID NO: 40; residues 572-1117 of SEQ ID NO: 41; residues 341-655 of SEQ ID NO: 42; residues 341-655 of SEQ ID NO: 43; residues 342- 657 of SEQ ID NO:
  • chimeric IFNG receptors are comprised of a first chimeric polypeptide that includes an IFNGR1 extracellular domain, a first transmembrane domain, and a first intracellular signaling domain.
  • the first intracellular signaling domain is a heterologous signaling domain of a cytokine receptor.
  • the first intracellular signaling domain is a modified IFNGR1 intracellular signaling domain.
  • chimeric IFNG receptors that include a first chimeric polypeptide as described above and further include a second chimeric polypeptide that includes an IFNGR2 extracellular domain, a second transmembrane domain, and a second intracellular signaling domain.
  • the second intracellular signaling domain is a heterologous signaling domain of a cytokine receptor. In other aspects, the second intracellular signaling domain is an IFNGR2 intracellular signaling domain, for example a wild-type IFNGR2 intracellular signaling domain.
  • the first cytokine receptor and the second cytokine receptor are individually selected from the group consisting of EPOR, GHR, TPOR, PRLR, LEPR, TSLPR, CSF2RB, IL3RA, IL2RB, IL7R, IL9R, INSR, GP130, CD28, 4-1BB, ICOS, CD2, TLR1, TLR2, TLR4, TLR6, EGFR, IGF1R, MET, ROS1, GMCSFRA, IL2RG, IL6R, IL11RA, OSMRB, IL10RA, IL10RB, IL22R, IL20RA and IL20RB.
  • the amino acid sequence of the first intracellular signaling domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 269-503 of SEQ ID NO: 1; residues 269-433 of SEQ ID NO: 2; residues 270- 619 of SEQ ID NO: 3; residues 268-389 of SEQ ID NO: 4; residues 270-633 of SEQ ID NO: 5; residues 269-571 of SEQ ID NO: 6; residues 267-385 of SEQ ID NO: 7; residues 263-699 of SEQ ID NO: 8; residues 266-318 of SEQ ID NO: 9; residues 271-555 of SEQ ID NO: 10; residues 271-465 of SEQ ID NO: 11; residues 267-496 of SEQ ID NO: 12; residues 269-671 of SEQ ID NO: 13; residues 268-544 of SEQ ID NO: 14; residues 281-3
  • the amino acid sequence of the first intracellular signaling domain comprises or consists of residues 269-503 of SEQ ID NO: 1; residues 269-433 of SEQ ID NO: 2; residues 270-619 of SEQ ID NO: 3; residues 268-389 of SEQ ID NO: 4; residues 270- 633 of SEQ ID NO: 5; residues 269-571 of SEQ ID NO: 6; residues 267-385 of SEQ ID NO: 7; residues 263-699 of SEQ ID NO: 8; residues 266-318 of SEQ ID NO: 9; residues 271-555 of SEQ ID NO: 10; residues 271-465 of SEQ ID NO: 11; residues 267-496 of SEQ ID NO: 12; residues 269-671 of SEQ ID NO: 13; residues 268-544 of SEQ ID NO: 14; residues 281-313 of SEQ ID NO: 15; residues 273-313 of SEQ ID NO: 16; residues 2
  • the amino acid sequence of the second intracellular signaling domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 801-1035 of SEQ ID NO: 28; residues 731-895 of SEQ ID NO: 29; residues 918-1267 of SEQ ID NO: 30; residues 686-807 of SEQ ID NO: 31; residues 932- 1295 of SEQ ID NO: 32; residues 869-1171 of SEQ ID NO: 33; residues 610-1046 of SEQ ID NO: 34; residues 685-879 of SEQ ID NO: 35; residues 969-1371 of SEQ ID NO: 36; residues 1000-1053 of SEQ ID NO: 37; residues 851-936 of SEQ ID NO: 38; residues 761-846 of SEQ ID NO: 39; residues 792-877 of SEQ ID NO: 40; residues 841-1117
  • the amino acid sequence of the second intracellular signaling domain comprises or consists of residues 801- 1035 of SEQ ID NO: 28; residues 731-895 of SEQ ID NO: 29; residues 918-1267 of SEQ ID NO: 30; residues 686-807 of SEQ ID NO: 31; residues 932-1295 of SEQ ID NO: 32; residues 869-1171 of SEQ ID NO: 33; residues 610-1046 of SEQ ID NO: 34; residues 685-879 of SEQ ID NO: 35; residues 969-1371 of SEQ ID NO: 36; residues 1000-1053 of SEQ ID NO: 37; residues 851-936 of SEQ ID NO: 38; residues 761- 846 of SEQ ID NO: 39; residues 792-877 of SEQ ID NO: 40; residues 841-1117 of SEQ ID NO: 41; residues 615-655 of SEQ ID NO: 42; residues 615-655 of SEQ ID NO: 43; residues 616-657 of SEQ
  • the first intracellular signaling domain is a modified IFNGR1 signaling domain; and/or the first intracellular signaling domain is a modified IFNGR1 6727-109858-02 signaling domain and the second intracellular signaling domain is a wild-type IFNGR2 signaling domain.
  • the amino acid sequence of the modified IFNGR1 intracellular signaling domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 267-489 of SEQ ID NO: 68; residues 267-489 of SEQ ID NO: 69; residues 267-472 of SEQ ID NO: 70; residues 267-489 of SEQ ID NO: 71; residues 267-497 of SEQ ID NO: 72; or residues 267-489 of SEQ ID NO: 73.
  • the amino acid sequence of the modified IFNGR1 intracellular signaling domain comprises or consists of residues 267-489 of SEQ ID NO: 68; residues 267- 489 of SEQ ID NO: 69; residues 267-472 of SEQ ID NO: 70; residues 267-489 of SEQ ID NO: 71; residues 267-497 of SEQ ID NO: 72; or residues 267-489 of SEQ ID NO: 73.
  • the wild-type IFNGR2 intracellular signaling domain includes residues 288-356 of SEQ ID NO: 148.
  • the transmembrane domain of the first chimeric polypeptide is a heterologous transmembrane domain (a transmembrane domain that is not from IFNGR1), such as a transmembrane domain from the same cytokine receptor as the first intracellular signaling domain
  • the transmembrane domain of the second chimeric polypeptide is a heterologous transmembrane domain (a transmembrane domain that is not from IFNGR2), such as a transmembrane domain from the same cytokine receptor as the second intracellular signaling domain.
  • the transmembrane domain of the first chimeric polypeptide is an IFNGR1 transmembrane domain and/or the transmembrane domain of the second chimeric polypeptide is an IFNGR2 transmembrane domain.
  • the amino acid sequence of the first chimeric polypeptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO:
  • the amino acid sequence of the first chimeric polypeptide comprises or consists of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; residues 1-503 of SEQ ID NO: 28; residues 1-433 of SEQ ID NO: 29; residues 1-619 of SEQ ID NO:
  • the amino acid sequence of the second chimeric polypeptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to residues 531-1035 of SEQ ID NO: 28; residues 461-895 of SEQ ID NO: 29; residues 645-1297 of SEQ ID NO: 30; residues 417-807 of SEQ ID NO: 31; residues 661-1295 of SEQ ID NO: 32; residues 599-1171 of SEQ ID NO: 33; residues 346-1046 of SEQ ID NO: 34; residues 413- 879 of SEQ ID NO: 35; residues 699-1371 of SEQ ID NO: 36; residues 727-1053 of SEQ ID NO: 37; residues 535-936 of SEQ ID NO: 38; residues 493-846 of SEQ ID NO: 39; residues 524-877 of SEQ ID NO: 40; residues 572-1
  • the amino acid sequence of the second chimeric polypeptide comprises or consists of residues 531-1035 of SEQ ID NO: 28; residues 461-895 of SEQ ID NO: 29; residues 645-1297 of SEQ ID NO: 30; residues 417-807 of SEQ ID NO: 31; residues 661-1295 of SEQ ID NO: 32; residues 599-1171 of SEQ ID NO: 33; residues 346-1046 of SEQ ID NO: 34; residues 413-879 of SEQ ID NO: 35; residues 699-1371 of SEQ ID NO: 36; residues 727-1053 of SEQ ID NO: 37; residues 535-936 of SEQ ID NO: 38; residues 493-846 of SEQ ID NO: 39; residues 524-877 of SEQ ID NO: 40; residues 572-1117 of SEQ ID NO: 41; residues 341-655 of SEQ ID NO: 42; residues 341-655 of SEQ ID NO: 43; residues 342- 657 of SEQ ID NO:
  • compositions that include a nucleic acid molecule(s), modified immune cell or chimeric IFNG receptor disclosed herein and a pharmaceutically acceptable carrier.
  • methods of treating cancer in a subject includes administering to the subject a therapeutically effective amount of a nucleic acid molecule(s), modified immune cell, chimeric IFNG receptor, or pharmaceutical composition disclosed herein.
  • the method further includes administering one or more additional anti-cancer therapies to the subject. IV.
  • IFNGR1 Chimeric Polypeptide Sequences Provided below are the amino acid sequences of exemplary chimeric polypeptides that include, in the N-terminal to C-terminal direction, an extracellular domain from IFNGR1, a transmembrane domain (from IFNGR1 or another cytokine receptor), and an intracellular signaling domain from a heterologous cytokine receptor (or a modified IFNGR1 signaling domain).
  • the amino acid sequences of the chimeric IFNGR1 polypeptides listed below the underlined portion of each sequence indicates the transmembrane 6727-109858-02 domain.
  • the tables below each sequence specify the amino acid residues that correspond to each domain of the chimeric polypeptide.
  • IFNGR1-EPORtm SEQ ID NO: 1
  • IFNGR1-IFNGR2 Chimeric Polypeptide Sequences Provided below are the amino acid sequences of exemplary chimeric polypeptides that include, in the N-terminal to C-terminal direction, a first extracellular domain from IFNGR1, a first transmembrane 6727-109858-02 domain (from IFNGR1 or another cytokine receptor), a first intracellular signaling domain from a heterologous cytokine receptor (or a modified IFNGR1 signaling domain), a 2A peptide sequence, a second extracellular domain from IFNGR2, a second transmembrane domain (from IFNGR2 or another cytokine receptor), and a second intracellular signaling domain from a heterologous cytokine receptor (or from IFNGR2).
  • the 2A peptide sequence upon expression of the chimeric polypeptide in a host cell, two polypeptide chains are produced – one that includes the first extracellular domain, the first transmembrane domain, and the first intracellular signaling domain, and another that includes the second extracellular domain, the second transmembrane domain, and the second intracellular signaling domain.
  • the underlined portions of each sequence indicate the transmembrane domains and italicized residues indicate a 2A sequence.
  • the 2A sequence includes, in the N-terminal to C-terminal direction, a furin cleavage site (RAKR), a spacer (SGSG), and the P2A sequence (ATNFSLLKQAGDVEENPGP).
  • RAKR furin cleavage site
  • SGSG spacer
  • ATNFSLLKQAGDVEENPGP P2A sequence
  • IFNGR1-2-EPORtm SEQ ID NO: 28
  • nucleic Acid Sequences Encoding IFNGR Chimeric Polypeptides Provided below are exemplary codon-optimized nucleic acid sequences encoding chimeric IFNGR1 polypeptides that include, in the N-terminal to C-terminal direction, an extracellular domain from IFNGR1, a transmembrane domain (from IFNGR1 or another cytokine receptor), and an intracellular signaling domain from a heterologous cytokine receptor (or a modified IFNGR1 signaling domain).
  • IFNGR1-EPORtm ATGGCGCTGCTGTTCCTCCTTCCTCTGGTGATGCAGGGCGTTAGCAGAGCCGAAATGGGAACAGCCGACCTCGGACCTAG CAGCGTTCCAACACCTACAAACGTGACCATCGAGAGCTACAACATGAACCCCATCGTGTACTGGGAGTACCAAATCATGC CTCAGGTGCCCGTGTTCACCGTGGAAGTCAAGAATTATGGCGTGAAGAACAGCGAGTGGATCGACGCTTGTATCAACATC AGCCACCACTACTGCAACATCAGCGATCACGTGGGAGATCCCAGCAACAGCCTGTGGGTGCGGGTGAAGGCTAGAGTGGG CCAAAAGGAGTCTGCCTACGCCAAGAGCGAAGAGTTCGCCGTGTGCAGAGATGGCAAGATCGGGCCTCCAAAGCTGGACA TCAGAAAGGAAGAAGCAGATGGCAAGATCGGGCCTCCAAAGCTGGACA TCAGAAAGGAAGAAGCAGATGGCAAGATCGGGCCTCCAAAGCTGGACA
  • nucleic acid molecules encoding both chimeric IFNGR1 and chimeric IFNGR2 polypeptides are exemplified below, the chimeric IFNGR1 polypeptide and the chimeric IFNGR2 polypeptide can be encoded by separate nucleic acid molecules.
  • IFNGR1 interferon gamma receptor 1
  • IFNGR2 interferon gamma receptor 2
  • Aspect 4 The nucleic acid molecule or molecules of aspect 3, wherein the first cytokine receptor and the second cytokine receptor are individually selected from the group consisting of: erythropoietin receptor (EPOR), growth hormone receptor (GHR), thrombopoietin receptor (TPOR), prolactin receptor (PRLR), leptin receptor (LEPR), thymic stromal lymphopoietin receptor (TSLPR), colony stimulating factor 2 receptor subunit beta (CSF2RB), interleukin 3 receptor subunit alpha (IL3RA), interleukin 2 receptor subunit beta (IL2RB), interleukin 7 receptor (IL7R), interleukin 9 receptor (IL9R), insulin receptor (INSR), membrane glycoprotein 130 (GP130), CD28, 4-1BB, inducible T cell costimulator (ICOS), CD2, toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), toll-
  • Aspect 5 The nucleic acid molecule or molecules of any one of aspects 1-4, wherein the amino acid sequence of the first intracellular signaling domain is at least 90% identical to residues 269-503 of SEQ ID NO: 1; residues 269-433 of SEQ ID NO: 2; residues 270-619 of SEQ ID NO: 3; residues 268- 389 of SEQ ID NO: 4; residues 270-633 of SEQ ID NO: 5; residues 269-571 of SEQ ID NO: 6; residues 267-385 of SEQ ID NO: 7; residues 263-699 of SEQ ID NO: 8; residues 266-318 of SEQ ID NO: 9; residues 271-555 of SEQ ID NO: 10; residues 271-465 of SEQ ID NO: 11; residues 267-496 of SEQ ID NO: 12; residues 269-671 of SEQ ID NO: 13; residues 268-544 of SEQ ID NO: 14; residues 281-313 of SEQ ID NO: 15; residues 273-313 of
  • Aspect 6 The nucleic acid molecule or molecules of any one of aspects 1-5, wherein the amino acid sequence of the first intracellular signaling domain comprises or consists of residues 269-503 of SEQ ID NO: 1; residues 269-433 of SEQ ID NO: 2; residues 270-619 of SEQ ID NO: 3; residues 268-389 of SEQ ID NO: 4; residues 270-633 of SEQ ID NO: 5; residues 269-571 of SEQ ID NO: 6; residues 267- 385 of SEQ ID NO: 7; residues 263-699 of SEQ ID NO: 8; residues 266-318 of SEQ ID NO: 9; residues 271-555 of SEQ ID NO: 10; residues 271-465 of SEQ ID NO: 11; residues 267-496 of SEQ ID NO: 12; residues 269-671 of SEQ ID NO: 13; residues 268-544 of SEQ ID NO: 14; residues 281-313 of SEQ ID NO: 15; residues 273-313 of S
  • Aspect 7 The nucleic acid molecule or molecules of any one of aspects 1-6, wherein the amino acid sequence of the second intracellular signaling domain is at least 90% identical to residues 801- 1035 of SEQ ID NO: 28; residues 731-895 of SEQ ID NO: 29; residues 918-1267 of SEQ ID NO: 30; residues 686-807 of SEQ ID NO: 31; residues 932-1295 of SEQ ID NO: 32; residues 869-1171 of SEQ ID NO: 33; residues 610-1046 of SEQ ID NO: 34; residues 685-879 of SEQ ID NO: 35; residues 969-1371 of SEQ ID NO: 36; residues 1000-1053 of SEQ ID NO: 37; residues 851-936 of SEQ ID NO: 38; residues 761- 846 of SEQ ID NO: 39; residues 792-877 of SEQ ID NO: 40; residues 841-1117 of SEQ ID NO: 41; residues 615-655 of SEQ ID NO: 42; residues 6
  • Aspect 8 The nucleic acid molecule or molecules of any one of aspects 1-7, wherein the amino acid sequence of the second intracellular signaling domain comprises or consists of residues 801- 1035 of SEQ ID NO: 28; residues 731-895 of SEQ ID NO: 29; residues 918-1267 of SEQ ID NO: 30; residues 686-807 of SEQ ID NO: 31; residues 932-1295 of SEQ ID NO: 32; residues 869-1171 of SEQ ID NO: 33; residues 610-1046 of SEQ ID NO: 34; residues 685-879 of SEQ ID NO: 35; residues 969-1371 of SEQ ID NO: 36; residues 1000-1053 of SEQ ID NO: 37; residues 851-936 of SEQ ID NO: 38; residues 761- 846 of SEQ ID NO: 39; residues 792-877 of SEQ ID NO: 40; residues 841-1117 of SEQ ID NO: 41; residues 615-655 of SEQ ID NO: 42; residues 615
  • Aspect 9 The nucleic acid molecule or molecules of aspect 1, wherein the first intracellular signaling domain is a modified IFNGR1 signaling domain.
  • Aspect 10 The nucleic acid molecule or molecules of aspect 2, wherein the first intracellular signaling domain is a modified IFNGR1 signaling domain and the second intracellular signaling domain is a wild-type IFNGR2 signaling domain.
  • Aspect 11 The nucleic acid molecule or molecules of aspect 1, wherein the first intracellular signaling domain is a modified IFNGR1 signaling domain.
  • the second intracellular signaling domain is a wild-type IFNGR2 signaling domain.
  • nucleic acid molecule or molecules of aspect 9 or aspect 10 wherein the amino acid sequence of the modified IFNGR1 intracellular signaling domain is at least 90% identical to residues 267-489 of SEQ ID NO: 68; residues 267-489 of SEQ ID NO: 69; residues 267-472 of SEQ ID NO: 70; residues 267-489 of SEQ ID NO: 71; residues 267-497 of SEQ ID NO: 72; or residues 267-489 of SEQ ID NO: 73.
  • amino acid sequence of the modified IFNGR1 intracellular signaling domain is at least 90% identical to residues 267-489 of SEQ ID NO: 68; residues 267-489 of SEQ ID NO: 69; residues 267-472 of SEQ ID NO: 70; residues 267-489 of SEQ ID NO: 71; residues 267-497 of SEQ ID NO: 72; or residues 267-489 of SEQ ID NO: 73.
  • nucleic acid molecule or molecules of any one of aspects 9-11 wherein the amino acid sequence of the modified IFNGR1 intracellular signaling domain comprises or consists of residues 267-489 of SEQ ID NO: 68; residues 267-489 of SEQ ID NO: 69; residues 267-472 of SEQ ID NO: 70; residues 267-489 of SEQ ID NO: 71; residues 267-497 of SEQ ID NO: 72; or residues 267-489 of SEQ ID NO: 73.
  • Aspect 13 The nucleic acid molecule or molecules of any one of aspects 10-12, wherein the wild-type IFNGR2 intracellular signaling domain comprises residues 288-356 of SEQ ID NO: 148.
  • Aspect 14 The nucleic acid molecule or molecules of any one of aspects 9-11, wherein the amino acid sequence of the modified IFNGR1 intracellular signaling domain comprises or consists of residues 267-489 of SEQ ID NO: 68; residues 267-489 of SEQ ID NO: 69
  • Aspect 15 The nucleic acid molecule or molecules of any one of aspects 2-14, wherein the second transmembrane domain is a second heterologous transmembrane domain.
  • Aspect 16 The nucleic acid molecule or molecules of aspect 14 or aspect 15, wherein: the first heterologous transmembrane domain is from the same cytokine receptor as the first intracellular signaling domain; and/or the second heterologous transmembrane domain is from the same cytokine receptor as the second intracellular signaling domain.
  • Aspect 18 The nucleic acid molecule or molecules of any one of aspects 2-13 and 17, wherein the second transmembrane domain is an IFNGR2 transmembrane domain.
  • Aspect 19 The nucleic acid molecule or molecules of any one of aspects 2-18, wherein the first chimeric polypeptide and the second chimeric polypeptide are encoded by a single nucleic acid molecule and the coding sequence for the first chimeric polypeptide and the coding sequence for the second chimeric polypeptide are separated by a nucleic acid sequence encoding a self-cleaving peptide.
  • Aspect 20 The nucleic acid molecule or molecules of aspect 19, wherein the self-cleaving peptide is a 2A peptide.
  • Aspect 21 The nucleic acid molecule or molecules of aspect 20, wherein the amino acid sequence of the self-cleaving peptide comprises residues 512-530 of SEQ ID NO: 28.
  • Aspect 22 The nucleic acid molecule or molecules of aspect 19, wherein the amino acid sequence of the self-cleaving peptide comprises residues 512-530 of SEQ ID NO: 28.
  • P2A porcine teschovirus-12A peptide
  • T2A a Thosea asigna virus 2A peptide
  • E2A equine rhinitis A virus 2A peptide
  • F2A foot and mouth disease virus 2A peptide
  • nucleic acid molecule or molecules of aspect 22 wherein the amino acid sequence of the P2A peptide comprises SEQ ID NO: 149, the amino acid sequence of the F2A peptide comprises SEQ ID NO: 150, the amino acid sequence of the E2A peptide comprises SEQ ID NO: 151, or the amino acid sequence of the T2A peptide comprises SEQ ID NO: 152.
  • Aspect 24 The nucleic acid molecule or molecules of any one of aspects 2-18, wherein the first chimeric polypeptide is encoded by a first nucleic acid molecule and the second chimeric polypeptide is encoded by a second nucleic acid molecule. Aspect 25.
  • the nucleic acid molecule or molecules of any one of aspects 1-24 which is codon- optimized for expression in human cells.
  • Aspect 26 The nucleic acid molecule or molecules of any one of aspects 1-25, comprising the nucleotide sequence of any one of SEQ ID NOs: 74-100, or a degenerate variant thereof.
  • Aspect 27 The nucleic acid molecule or molecules of any one of aspects 2-25, comprising the nucleotide sequence of any one of SEQ ID NOs: 101-146, or a degenerate variant thereof.
  • Aspect 28 The nucleic acid molecule or molecules of any one of aspects 1-27, operably linked to a promoter.
  • Aspect 29 The nucleic acid molecule or molecules of any one of aspects 1-27, operably linked to a promoter.
  • a vector comprising the nucleic acid molecule or molecules of any one of aspects 1- 28.
  • Aspect 30 The vector of aspect 29, which is a viral vector.
  • Aspect 31 The vector of aspect 30, which is a gammaretroviral vector or a lentiviral vector.
  • Aspect 32 A modified immune cell comprising the nucleic acid molecule or molecules of any one of aspects 1-28 or the vector of any one of aspects 29-31.
  • Aspect 33 The modified immune cell of aspect 32, wherein the immune cell is a T cell.
  • Aspect 34. The modified immune cell of aspect 33, wherein the T cell is a tumor-reactive T cell or a tumor-infiltrating lymphocyte (TIL).
  • TIL tumor-infiltrating lymphocyte
  • IFNGR1 interferon gamma receptor 1
  • the modified immune cell of aspect 36 further expressing a second chimeric polypeptide, wherein: the second chimeric polypeptide comprises an interferon gamma receptor 2 (IFNGR2) extracellular domain, a second transmembrane domain, and a second intracellular signaling domain, wherein the second intracellular signaling domain is a heterologous signaling domain of a cytokine receptor or an IFNGR2 intracellular signaling domain.
  • IFNGR2 interferon gamma receptor 2
  • the modified immune cell of aspect 38 wherein the first cytokine receptor and the second cytokine receptor are individually selected from the group consisting of: erythropoietin receptor (EPOR), growth hormone receptor (GHR), thrombopoietin receptor (TPOR), prolactin receptor (PRLR), leptin receptor (LEPR), thymic stromal lymphopoietin receptor (TSLPR), colony stimulating factor 2 receptor subunit beta (CSF2RB), interleukin 3 receptor subunit alpha (IL3RA), interleukin 2 receptor subunit beta (IL2RB), interleukin 7 receptor (IL7R), interleukin 9 receptor (IL9R), insulin receptor (INSR), membrane glycoprotein 130 (GP130), CD28, 4-1BB, inducible T cell costimulator (ICOS), CD2, toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), toll-like receptor 6(TLR6),
  • Aspect 40 The modified immune cell of any one of aspects 36-39, wherein the amino acid sequence of the first intracellular signaling domain is at least 90% identical to residues 269-503 of SEQ ID NO: 1; residues 269-433 of SEQ ID NO: 2; residues 270-619 of SEQ ID NO: 3; residues 268-389 of SEQ ID NO: 4; residues 270-633 of SEQ ID NO: 5; residues 269-571 of SEQ ID NO: 6; residues 267-385 of SEQ ID NO: 7; residues 263-699 of SEQ ID NO: 8; residues 266-318 of SEQ ID NO: 9; residues 271-555 of SEQ ID NO: 10; residues 271-465 of SEQ ID NO: 11; residues 267-496 of SEQ ID NO: 12; residues 269-671 of SEQ ID NO: 13; residues 268-544 of SEQ ID NO: 14; residues 281-313 of SEQ ID NO: 15; residues 273-313 of SEQ ID NO:
  • Aspect 41 The modified immune cell of any one of aspects 36-40 wherein the amino acid sequence of the first intracellular signaling domain comprises or consists of residues 269-503 of SEQ ID NO: 1; residues 269-433 of SEQ ID NO: 2; residues 270-619 of SEQ ID NO: 3; residues 268-389 of SEQ ID NO: 4; residues 270-633 of SEQ ID NO: 5; residues 269-571 of SEQ ID NO: 6; residues 267-385 of SEQ ID NO: 7; residues 263-699 of SEQ ID NO: 8; residues 266-318 of SEQ ID NO: 9; residues 271-555 of SEQ ID NO: 10; residues 271-465 of SEQ ID NO: 11; residues 267-496 of SEQ ID NO: 12; residues 269-671 of SEQ ID NO: 13; residues 268-544 of SEQ ID NO: 14; residues 281-313 of SEQ ID NO: 15; residues 273-313 of SEQ ID NO: 16; residue
  • Aspect 42 The modified immune cell of any one of aspects 36-41, wherein the amino acid sequence of the second intracellular signaling domain is at least 90% identical to residues 801-1035 of SEQ ID NO: 28; residues 731-895 of SEQ ID NO: 29; residues 918-1267 of SEQ ID NO: 30; residues 686-807 of SEQ ID NO: 31; residues 932-1295 of SEQ ID NO: 32; residues 869-1171 of SEQ ID NO: 33; residues 610- 1046 of SEQ ID NO: 34; residues 685-879 of SEQ ID NO: 35; residues 969-1371 of SEQ ID NO: 36; residues 1000-1053 of SEQ ID NO: 37; residues 851-936 of SEQ ID NO: 38; residues 761-846 of SEQ ID NO: 39; residues 792-877 of SEQ ID NO: 40; residues 841-1117 of SEQ ID NO: 41; residues 615-655 of SEQ ID NO: 42; residues 615-655 of S
  • Aspect 43 The modified immune cell of any one of aspects 36-42, wherein the amino acid sequence of the second intracellular signaling domain comprises or consists of residues 801-1035 of SEQ ID NO: 28; residues 731-895 of SEQ ID NO: 29; residues 918-1267 of SEQ ID NO: 30; residues 686-807 of SEQ ID NO: 31; residues 932-1295 of SEQ ID NO: 32; residues 869-1171 of SEQ ID NO: 33; residues 610- 1046 of SEQ ID NO: 34; residues 685-879 of SEQ ID NO: 35; residues 969-1371 of SEQ ID NO: 36; residues 1000-1053 of SEQ ID NO: 37; residues 851-936 of SEQ ID NO: 38; residues 761-846 of SEQ ID NO: 39; residues 792-877 of SEQ ID NO: 40; residues 841-1117 of SEQ ID NO: 41; residues 615-655 of SEQ ID NO: 42; residues 615-655 of SEQ
  • Aspect 44 The modified immune cell of aspect 36, wherein the first intracellular signaling domain is a modified IFNGR1 signaling domain.
  • Aspect 45 The modified immune cell of aspect 37, wherein the first intracellular signaling domain is a modified IFNGR1 signaling domain and the second intracellular signaling domain is a wild-type IFNGR2 signaling domain.
  • the modified immune cell of aspect 44 or aspect 45 wherein the amino acid sequence of the modified IFNGR1 intracellular signaling domain is at least 90% identical to residues 267- 489 of SEQ ID NO: 68; residues 267-489 of SEQ ID NO: 69; residues 267-472 of SEQ ID NO: 70; residues 267-489 of SEQ ID NO: 71; residues 267-497 of SEQ ID NO: 72; or residues 267-489 of SEQ ID NO: 73.
  • Aspect 47 Aspect 47.
  • Aspect 48 The modified immune cell of any one of aspects 45-47, wherein the wild-type IFNGR2 intracellular signaling domain comprises residues 288-356 of SEQ ID NO: 148.
  • Aspect 55 The modified immune cell of any one of aspects 36-54, wherein the amino acid sequence of the first chimeric polypeptide comprises or consists of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; residues 1-503 of SEQ ID NO: 28; residues 1-433 of SEQ ID
  • Aspect 56 The modified immune cell of any one of aspects 37-55, wherein the amino acid sequence of the second chimeric polypeptide is at least 90% identical to residues 531-1035 of SEQ ID NO: 6727-109858-02 28; residues 461-895 of SEQ ID NO: 29; residues 645-1297 of SEQ ID NO: 30; residues 417-807 of SEQ ID NO: 31; residues 661-1295 of SEQ ID NO: 32; residues 599-1171 of SEQ ID NO: 33; residues 346-1046 of SEQ ID NO: 34; residues 413-879 of SEQ ID NO: 35; residues 699-1371 of SEQ ID NO: 36; residues 727-1053 of SEQ ID NO: 37; residues 535-936 of SEQ ID NO: 38; residues 493-846 of SEQ ID NO: 39; residues 524-877 of SEQ ID NO: 40; residues 572-1117 of SEQ ID NO: 41; residues 341-655 of SEQ ID NO: 42; residue
  • Aspect 57 The modified immune cell of any one of aspects 37-56, wherein the amino acid sequence of the second chimeric polypeptide comprises or consists of residues 531-1035 of SEQ ID NO: 28; residues 461-895 of SEQ ID NO: 29; residues 645-1297 of SEQ ID NO: 30; residues 417-807 of SEQ ID NO: 31; residues 661-1295 of SEQ ID NO: 32; residues 599-1171 of SEQ ID NO: 33; residues 346-1046 of SEQ ID NO: 34; residues 413-879 of SEQ ID NO: 35; residues 699-1371 of SEQ ID NO: 36; residues 727- 1053 of SEQ ID NO: 37; residues 535-936 of SEQ ID NO: 38; residues 493-846 of SEQ ID NO: 39; residues 524-877 of SEQ ID NO: 40; residues 572-1117 of SEQ ID NO: 41; residues 341-655 of SEQ ID NO: 42; residues 341-655 of SEQ
  • a chimeric interferon gamma receptor comprising a first chimeric polypeptide, wherein the first chimeric polypeptide comprises an interferon gamma receptor 1 (IFNGR1) extracellular domain, a first transmembrane domain, and a first intracellular signaling domain, wherein the first intracellular signaling domain is a heterologous signaling domain of a cytokine receptor or a modified IFNGR1 intracellular signaling domain.
  • IFNGR1 interferon gamma receptor 1
  • the chimeric IFNGR of aspect 58 further comprising a second chimeric polypeptide, wherein the second chimeric polypeptide comprises an interferon gamma receptor 2 (IFNGR2) extracellular domain, a second transmembrane domain, and a second intracellular signaling domain, wherein the second intracellular signaling domain is a heterologous signaling domain of a cytokine receptor or an IFNGR2 intracellular signaling domain.
  • IFNGR2 interferon gamma receptor 2
  • the chimeric IFNGR of aspect 58 or aspect 59 wherein the first intracellular signaling domain is a heterologous intracellular signaling domain of a first cytokine receptor and/or the second intracellular signaling domain is a heterologous intracellular signaling domain of a second cytokine receptor.
  • the first intracellular signaling domain is a heterologous intracellular signaling domain of a first cytokine receptor and/or the second intracellular signaling domain is a heterologous intracellular signaling domain of a second cytokine receptor.
  • the chimeric IFNGR of aspect 60 wherein the first cytokine receptor and the second cytokine receptor are individually selected from the group consisting of: erythropoietin receptor (EPOR), growth hormone receptor (GHR), thrombopoietin receptor (TPOR), prolactin receptor (PRLR), leptin receptor (LEPR), thymic stromal lymphopoietin receptor (TSLPR), colony stimulating factor 2 receptor subunit beta (CSF2RB), interleukin 3 receptor subunit alpha (IL3RA), interleukin 2 receptor subunit beta (IL2RB), interleukin 7 receptor (IL7R), interleukin 9 receptor (IL9R), insulin receptor (INSR), membrane glycoprotein 130 (GP130), CD28, 4-1BB, inducible T cell costimulator (ICOS), CD2, toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), toll-like receptor 6(T
  • Aspect 62 The chimeric IFNGR of aspect 58 wherein the first intracellular signaling domain is a modified IFNGR1 signaling domain.
  • Aspect 63 The chimeric IFNGR of aspect 59, wherein the first intracellular signaling domain is a modified IFNGR1 signaling domain and the second intracellular signaling domain is a wild-type IFNGR2 signaling domain.
  • Aspect 64 The chimeric IFNGR of any one of aspects 58-63, wherein the first transmembrane domain is a first heterologous transmembrane domain and/or the second transmembrane domain is a second heterologous transmembrane domain. 6727-109858-02 Aspect 65.
  • Aspect 66 A pharmaceutical composition comprising the modified immune cell of any one of aspects 32-57 and a pharmaceutically acceptable carrier.
  • Aspect 67. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the modified immune cell of any one of aspects 32-57 or the pharmaceutical composition of aspect 66.
  • Aspect 68. The method of aspect 67, further comprising administering one or more additional anti-cancer therapies to the subject.
  • Example 1 Design of chimeric IFNG receptors
  • the native IFNGR is comprised of two homodimers of IFNGR1 and IFNGR2 that are activated upon binding to IFN- ⁇ .
  • the chimeric IFNG receptors disclosed herein include extracellular domains from the IFNGR fused to cytoplasmic signaling domains from other receptors that can activate pathways that enhance T cell activity.
  • Entire cytoplasmic domains (see, e.g., schematics ii, iii and iv in FIG.1 and SEQ ID NOs: 1-67) or parts of cytoplasmic domains (see, e.g., schematic v in FIG.1 and SEQ ID NOs: 68-73) from other receptors can be used.
  • the transmembrane domain can be derived from any receptor (e.g., from IFNGR1, IFNGR2, or the signaling receptor).
  • the sequences were separated by a sequence encoding a 2A self-cleaving (ribosomal skipping) peptide (such as SEQ ID NO: 149).
  • Nucleic acid sequences encoding IFNG chimeric receptors were codon-optimized (e.g., SEQ ID NOs: 74-146), synthesized and cloned into the MSGV1 gammaretroviral plasmid (GenScript). Retroviral vector was generated by transfecting 293GP cells with the MSGV1 plasmid encoding the IFNG chimeric receptor and the envelope plasmid RD114. Supernatants (retroviral vector) were harvested about 2 days later and used to transduce human T cells.
  • Example 2 Generation and characterization of engineered T cells expressing chimeric IFNG receptors Human peripheral blood mononuclear cells were stimulated with anti-CD3 antibody OKT3 (50 ng/mL) and IL-2 (300 IU/ml) for two days and then transduced with a retroviral vector encoding a TCR recognizing an HLA-C*08:02-restricted KRAS-G12D neoantigen alone (FIG.2A), or co-transduced with either a retroviral vector encoding an IFNG chimeric receptor containing the IFNGR1 extracellular domain fused to the IL-2 receptor beta chain transmembrane and cytoplasmic domains and the IFNGR2 extracellular 6727-109858-02 domain fused to the common gamma chain transmembrane and cytoplasmic domains (IFNGR/IL2R) (FIG.
  • IFNGR/IL2R common gamma chain transmembrane and cytoplasm
  • IFNG GMCSF receptor alpha transmembrane and cytoplasmic domains
  • FIGS.2A-2C show the evaluation of biochemical signaling capacity of T cells expressing the exemplary IFNG chimeric receptors (IFNGR/IL2R or IFNGR/GMCSFR) after IFNG stimulation as measured by phosphorylated STAT5 (P-STAT5) by flow cytometry.
  • IFNGR/IL2R or IFNGR/GMCSFR exemplary IFNG chimeric receptors
  • Example 3 In vitro cytotoxicity of chimeric IFNG receptors against a pancreatic cancer cell line T cells were transduced to express the KRAS G12D-reactive TCR alone, or co-transduced to express either an IFNG chimeric receptor containing the IFNGR1 extracellular domain fused to the IL-2 receptor beta chain transmembrane and cytoplasmic domains and the IFNGR2 extracellular domain fused to the common gamma chain transmembrane and cytoplasmic domains (IFNGR/IL2R), or an IFNG receptor containing the IFNGR1 extracellular domain fused to the common beta chain cytoplasmic and transmembrane domains and the IFNGR2 extracellular domain fused to the GMCSF receptor alpha transmembrane and cytoplasmic domains (IFNGR/GMCSFR).
  • IFNGR/GMCSFR GMCSF receptor alpha transmembrane and cytoplasmic domains
  • FIG.3A shows GFP fluorescence (as a measure of tumor cell growth) of the KRAS-G12D-positive and HLA-C*08:02- positive HPAC-GFP pancreatic cancer cell line in the presence of T cells transduced to express the HLA- C*08:02-restricted KRAS-G12D reactive TCR with or without 300 IU/ml IL-2.
  • FIGS.3B-3C show GFP fluorescence of the KRAS-G12D-positive and HLA-C*08:02-positive HPAC-GFP pancreatic cancer cell line in the presence of T cells co-transduced to express the HLA-C*08:02-restricted KRAS-G12D reactive TCR and IFNGR/IL2R chimeric receptors (FIG.3B) or IFNGR/GMCSFR chimeric receptors (FIG.3C). No exogenous cytokines were added to the coculture.
  • the data from FIG.3A were incorporated into FIG.3B and FIG.3C to facilitate comparison against the control TCR alone without or with 300 IU/ml IL-2.

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Abstract

L'invention concerne des récepteurs d'interféron gamma (IFNG) chimériques qui comprennent le domaine extracellulaire du récepteur IFNG 1 (IFNGR1) et/ou du récepteur IFNG 2 (IFNGR2) fusionné aux domaines intracellulaires de récepteurs de cytokine hétérologues. Lors de l'activation de cellules immunitaires (telles que des lymphocytes T) exprimant les récepteurs chimériques, les cellules immunitaires sécrètent l'IFN-g, qui se lie aux récepteurs chimériques IFNG et transmet des signaux qui améliorent la prolifération et/ou la fonction des cellules immunitaires modifiées. De telles cellules immunitaires modifiées peuvent être utilisées, par exemple, pour améliorer l'immunothérapie anticancéreuse.
PCT/US2024/049981 2023-10-06 2024-10-04 Récepteurs d'interféron-gamma (ifn-g) modifiés (ifngr) pour améliorer l'immunothérapie cellulaire Pending WO2025076367A1 (fr)

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WO2021050752A1 (fr) * 2019-09-11 2021-03-18 The Board Of Trustees Of The Leland Stanford Junior University Protéines réceptrices orthogonales chimériques et leurs méthodes d'utilisation
US20220162288A1 (en) * 2020-11-25 2022-05-26 Catamaran Bio, Inc. Cellular therapeutics engineered with signal modulators and methods of use thereof
US20230159892A1 (en) * 2020-04-06 2023-05-25 Synthekine, Inc. Engineered immune cells
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US20190241633A1 (en) * 2016-05-04 2019-08-08 Curevac Ag Rna encoding a therapeutic protein
WO2021050752A1 (fr) * 2019-09-11 2021-03-18 The Board Of Trustees Of The Leland Stanford Junior University Protéines réceptrices orthogonales chimériques et leurs méthodes d'utilisation
US20230159892A1 (en) * 2020-04-06 2023-05-25 Synthekine, Inc. Engineered immune cells
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WO2023177206A1 (fr) * 2022-03-17 2023-09-21 연세대학교 산학협력단 Composition permettant le diagnostic du cancer du pancréas

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